The present invention relates to methods and apparatus for the manipulation of proteins and peptides by reversibly binding proteins and peptides to beads having a coating capable of binding with native proteins and peptides. More particularly, the invention is directed to methods and apparatus for capturing native proteins and peptides on magnetically responsive beads.
Magnetically responsive beads or particles of small sizes are well known and have been used for a number of separation and diagnostic purposes. Small magnetic beads have been used to bind to cells and other particles in order to manipulate and separate them. See, for example, U.S. Pat. Nos. 4,230,685, 3,970,518, 5,508,164, 5,567,326 and 4,018,886.
Magnetic beads have been prepared from a variety of polymers with metal particles therein, including those prepared by gelatin, colloidal metal and other agents. An example of this type of bead is disclosed in U.S. Pat. No. 4,582,622. Chemical coupling of proteins to reactive moieties on magnetic beads has been proposed using various linking groups as disclosed in U.S. Pat. No. 4,628,037. While certain bonds may be cleavable, the protein binding is not disclosed as being reversible.
Magnetic beads coated with streptavidin have been used for immobilizing biotinylated substances. Magnetic beads of a polysaccharide (dextran) with a cleavable linker to protein A to immobilize antibodies and bind to a protein have been proposed in U.S. Pat. No. 4,452,773. Protein A coated magnetic beads have also been proposed in U.S. Pat. No. 5,158,871. U.S. Pat. No. 4,297,337 discloses magnetic beads made of porous glass.
U.S. Pat. No. 5,900,481 to Lough et al discloses using coated beads to bind to DNA and thereby manipulate the DNA. Other disclosures of manipulating various molecules bound to beads include Girault et al, Analytical Chemistry 68(13): 2122-6 (1996), and Tang et al, Nucleic Acids Research 23(16): 3126-31 (1995).
A number of magnetic separators for small beads are commercially available, which can readily remove micron size ferromagnetic particles from solution by employing relatively inexpensive permanent magnets. Examples of such magnetic separators include those manufactured by Ciba-Corning Medical Diagnostics, Wampole, Mass., the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, Mass., U.S.A., the DYNAL MPC-1 manufactured by DYNAL, Inc., Great Neck, N.Y., U.S.A., and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Mass., U.S.A.
U.S. Pat. No. 5,834,197 to Parton discloses a method of capturing a species from a liquid using coated magnetic beads. The beads have a selective affinity for an antigen. A labeled antibody is added to sandwich the antigen and provide a detectable label bound to the magnetic beads for easy detection and recovery of the antigen.
One method for separation and purification of many different proteins in a sample is by using two-dimensional gel electrophoresis. Electrophoreses separation procedures are routinely applied to complex mixtures of proteins to resolve individual molecular species. Two-dimensional electrophoresis utilizes two orthogonal separations to produce a highly resolved pattern of protein spots, many of which may be effectively homogeneous protein samples. Examples include: O""Farrel, J. Biol. Chem. 250: 4007-4021, (1975), Anderson, et al, Anal. Biochem. 85: 3311-340, (1978), Anderson, et al, Anal. Biochem. 85: 341-354, (1978), Anderson, et al, Anal. Biochem. 93: 312-320, (1979) and Giometti, et, al, Anal. Biochem. 102: 47-58, (1980).
Methods have been described for the recovery of isolated proteins from such spots, generally for the purpose of characterization by microchemical Edman sequencing, amino acid analysis or mass spectrometry, or for preparation of an antigen for the immunization of animals to produce antisera.
It is current practice to identify an isolated protein found in these gels by excising a spot containing the protein of interest. Trypsin is added for cleaving the protein to generate peptides that are then identified by mass spectrometry (MS). One example is disclosed in Rosenfeld et al, Analytical Biochemistry 203:173-179 (1992). From the pattern of peptide molecular weights, or their fragments, one can then deduce the identity of the original protein by comparisons against a suitable sequence database.
One of the difficulties of this practice is that the quantity of these peptides is small and they diffuse freely from the gel into the surrounding liquid and interact with the walls of the container. The peptides in dilute solutions are recovered for use in MS analysis by centrifugal vacuum concentration (Speedvac system) or by lyophilization of a small volume of the surrounding liquid. This recovery step is fraught with difficulties, as the dilute peptides are exposed to plastic and other surfaces upon which they may be captured and thus lost to the investigator.
Likewise, when one wishes to isolate an intact protein from a spot on the gel, a recovery method is used. One method is electroelution, in which the protein is caused to move out of the excised gel spot under the influence of an electric field. In most procedures, the eluted protein is recovered in a small fluid volume by electrophoresing it against a membrane that is impermeable to protein of the expected molecular mass. Devices designed to accomplish this procedure are sold by BioRad, Pharmacia Biotech, Millipore and others. Such techniques suffer from low quantitative recovery probably due to the imperfect recovery of protein off the barrier membrane.
Other published procedures include adsorbing proteins into small C18 reverse phase chromatography columns for the HP G1000A protein sequencer. The Hewlett Packard protein sequencer may use sample cartridges that are small two-part disposable columns with an upper half containing C18 chromatography support, and a lower half containing an ion exchange support. As different aqueous and organic solvents flow through the cartridge, the direction of flow can be arranged so that the applied protein or peptide is always immobilized on the xe2x80x9cdownstreamxe2x80x9d support to prevent loss of material. It has been attempted to apply the protein to the C18 half of the column by electroeluting gel spot protein through the column. However, gas bubbles frequently form in the column and block the flow of current. Once successfully immobilized, the protein is then chemically modified and degraded for Edman sequencing directly.
Another technique is electroblotting, in which a gel is sandwiched against a protein-binding membrane, and an electric field is applied using suitable electrodes and buffers to cause the proteins to migrate out of the gel and through the membrane, where they are then immobilized. One, such example using a nitrocellulose blotting sheet is disclosed in Towbin, et al, Proc. Nat. Acad. Sci. USA 1979, 76, 4350-4354. The gel-separated proteins retain their spatial relationships while they move into the membrane, so that the pattern of separated proteins bound to the membrane is generally the same as the pattern of proteins resolved in the gel. When used with proteins this procedure is generally called xe2x80x9cWestern blottingxe2x80x9d. A drawback of the blotting approach is the difficulty of subsequently removing the blotted proteins from the membrane for subsequent manipulation in a different format.
Polyvinylidene difluoride (PVDF) is very hydrophobic and binds proteins well. Polyvinylidene difluoride membranes (Immobilon(copyright), Milipore) have been used for recovery of proteins from two-dimensional gels and for using in situ digestion and transfer of peptides from two-dimensional gels. Kennedy et al, Proc. Natl. Acad. Sci. U.S.A. 85(18):7008-12 (1988) and Kamps et al, Anal. Biochem, 176(1):22-7 (1989). Membranes bearing charged groups have also been used (Millipore cationic membranes). In cases, where the resolved protein is small, or the gel medium has very large pores (as with agarose gels), it is possible to recover some of the protein by passive elution from the gel.
A common problem with the elution methods is that the protein solution is very dilute, not easily recovered in a small volume and in a form that is difficult to manipulate by robotic methods.
High-resolution t two-dimensional electrophoretic gels of human serum are also well known. Anderson, et al In: The Plasma Proteins, F. Putnam, ed., Academic Press, 2nd Ed., Vol. 4, pp 221-270, (1984).
Repeated adsorption and elution of antibodies wherein the immobilized antigen is used to eventually purify many times more of its specific antibody has been proposed. Anderson et al, Anal. Biochem. 66(1):159-74 (1975) and Anderson et al, Anal. Biochem. 68(2):371-93 (1975).
An object of the present invention is to provide small beads having a non-specific peptide-adsorptive surface, where beads can be readily manipulated for physical collection, movement and release by electromechanical means.
A further object of the present invention is to make and use small magnetically responsive beads having a reversible or irreversible attachment with a protein or peptide that in turn may be reversibly bound to an antibody or other receptor such that each reversible binding is elutable under different conditions than the protein or peptide to the bead.
Yet another object of the present invention is to provide a method and apparatus for the recovery and handling of proteins previously purified, such as those resolved as spots on 2-D electrophoretic gels by electroeluting protein from an excised gel spot through a bed of protein adsorbing beads.
Another object of the present invention is to attach an affinity label on a protein or peptide before purification or before separation from a permeable support material by reaction of the whole permeable support material containing the protein or peptide with reagents to link the affinity label to the protein or peptide.
In yet another embodiment, the affinity label bonded to the protein or peptide does not alter the net molecular charge and isoelectric point of the protein or peptide.
It is an object of the present invention to prepare proteins and peptides in a matrix for matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry or electrospray (ES) mass spectrometry, particularly for LC/MS/MS by elution from magnetically responsive beads.
It is still another object of the present invention to provide small amounts of antibodies having affinities to many specific proteins and to use bead immobilized proteins for affinity chromatography.
It is a further object of the present invention to prepare large amounts of antibodies and proteins from samples having low concentrations of the antibodies or proteins than can be prepared by analytical methods such as 2D electrophoresis or affinity capture in a single run.
It is another object of the present invention to provide a robotic assembly for manipulating magnetically responsive beads and to provide automated processing of many different proteins and antibodies, recovering the proteins or antibodies in parallel, and optionally identifying the proteins or associated components bound thereto in a complex.
It is another object of the present invention to provide a permanent magnet probe for the attraction and release of magnetically responsive beads where the magnet is moved within the probe to selectively attract and release the beads as a means to manipulate bound substances through plural reaction stations.
The present invention separates proteins by two-dimensional electrophoresis followed by excising a piece of gel containing a protein of interest, electro-eluting the protein of interest in a special apparatus, capturing the protein on magnetically responsive beads, manipulating the beads with a magnetic probe to wash, preferably by immersion, optionally renature, contact with antibody, elute the antibody, elute the protein, or analyze the protein for identification. Alternatively, the protein of interest is first cleaved and the peptides eluted and captured followed by washing and eluting directly onto a mass spectroscopy analyzer.
The invention accomplishes the manipulation of proteins and peptides in a manner where separation, purification, washing and elution occurs in large volumes of solution but the proteins or peptides being manipulated are quantitatively recoverable in small volumes of solution. The manipulation is performed by alternatively magnetizing and demagnetizing a probe after moving to another location.
The objects and advantages of the invention are basically attained by providing a magnetically responsive bead having a surface with an affinity binding member or a hydrophobic coating thereon. The coatings are capable of reversibly binding a protein or a peptide.
The objects of the invention also provide a coupling system for purifying a greater molar quantity of a binding partner which in turn may be cycled to purify a greater molar quantity of the original binding partner.
The foregoing objects and advantages of the invention are further attained by providing a bead composition comprising first magnetically responsive beads with a first coating having a binding affinity for immobilizing a first protein on the first beads; and second magnetically responsive beads with a second coating having a binding affinity for immobilizing a second protein on the second beads.
The foregoing objects and advantages of the invention are still further attained by providing a method of recovering proteins or peptides from a sample. The method comprises the steps of: providing a solution or dispersion of the sample; contacting the solution with an amount of magnetically responsive microbeads, and recovering the beads from the solution. The microbeads have an outer surface with a hydrophobic coating having a binding affinity for proteins or peptides. The microbeads are contacted with the solution for sufficient time to bind the proteins or peptides to the beads.
The foregoing objects and advantages of the invention are yet further attained by providing a method of recovering a target component from a solution comprising the steps of: providing an amount of magnetically responsive beads having a coating with a binding affinity for the target compound, contacting the beads with the solution for sufficient time to attach the target compound to the beads, separating the beads from the solution, and eluting the target component from the beads.
The foregoing objects and advantages of the invention are still further attained by providing a method of isolating a target component from a sample, comprising the steps of: contacting a solution or dispersion of the target component with a magnetically responsive microbead having a coating with a binding affinity for the target component, the contacting step being for a sufficient time to enable the target component to bind to the beads; activating a probe to induce an electromagnetic field to the dispersion and capturing the beads on the probe; transferring the probe and captured beads to a first washing liquid; and deactivating the probe to release the beads into the washing liquid to remove impurities and isolate the target component.
The foregoing objects and advantages of the invention are yet further attained by providing a method of analyzing a target component comprising the steps of: contacting a solution or dispersion of a target component with an amount of magnetically responsive microbeads having a coating with a binding affinity for the target component, aspirating the solution or dispersion through a tube having a filter device and collecting the beads on the filter device, positioning an end of the tube in an inlet of an analytical device; and eluting the target component from the beads directly into the analytical device and analyzing the target component. The contacting step is for sufficient time for the target component to bind to the beads.
The foregoing objects and advantages of the invention are further attained by providing an apparatus for recovering an amount of magnetically responsive beads from a liquid. The apparatus comprises a vessel for Containing a liquid having an amount of magnetically responsive beads; and a probe mounted for reciprocating movement into and out of the vessel. The probe is capable of selectively producing an electromagnetic field. The probe has a top end and a bottom end land a collar coupled to the top end.
The foregoing objects and advantages of the invention are still further attained by providing an apparatus for separating a target compound from a solution or dispersion. The apparatus comprises a first vessel for containing a first electrically conductive liquid; a second vessel for containing a second electrically conductive liquid, the second liquid containing the target compound; a first electrode positioned in the first vessel for making electrical contact with the first liquid; a second electrode positioned in the second vessel for making electrical contact with the second liquid; a porous member coupled to the second vessel for separating the first liquid from the second liquid while allowing an electric current to pass between the electrodes; an amount of magnetically responsive beads having a coating of material with an affinity for the target compound; the beads being positioned on the porous member, and an electrical power source for producing an electric current between the electrodes to cause the target compound to migrate toward the beads to bind with the coating material.
The foregoing objects and advantages of the invention are further attained by providing an apparatus for separating a target compound from a solution or dispersion. The apparatus comprises a hollow tube having a first open end and second open end, and has an axial passage between the first end and the second end; a porous filter member disposed in the axial passage; an aspirating device coupled to the second end of the tube; and a magnet for producing an electromagnetic field in the axial passage for capturing magnetically responsive beads.
The foregoing objects and advantages of the invention are further attained by providing an apparatus for transferring magnetically responsive beads. The apparatus comprises a first vessel having a closed bottom end and an open top end, the first vessel being dimensioned for containing a liquid containing an amount of magnetically responsive beads; a second vessel having a closed bottom end and an open top end, the open top end of the second vessel being in communication with the open top end of the first vessel; and a first magnet for capturing the beads from the first vessel and transferring the beads to the second vessel.